Blood samples often contain important hydrophobic components for which some quantitative or qualitative analysis is desired. Drugs, drug metabolites or degradation products, and lipophilic materials (e.g., cholesterol) are examples of such hydrophobic components found in whole blood.
It is well established that the therapeutic effect of a drug is related to the drug in its free form, not bound to blood proteins or cellular components, and not metabolized. To determine an effective therapeutic dose, therefore, it would be useful to have a process that enables quantitating the amount of a drug in the free form relative to that which is bound to blood components and that which has been metabolized.
In the prior art, high performance liquid chromatography (HPLC) has been used to perform this analysis. However, conventional HPLC columns used for this purpose require cells to be removed prior to analysis to avoid column blockage. When protein accumulation might cause column deterioration, protein separation prior to analysis is also required. Separation of these components (typically through filtration, centrifugation or solvent extraction) is time consuming and so limits rapid, time-dependent studies, during which time a new equilibrium may be established. This limitation is especially significant for such drugs as radiolabelled flow tracers; the biodeposition of these drugs depends upon initial first pass extraction, requiring drug analysis shortly after exposure to biological fluids.
A method described in the literature permits the direct injection of whole blood samples to an HPLC system (Tamai et al., J. Chromatography, 423 (1987), 147-168) This method requires the whole blood samples to pass through a pre-column containing a resin with particles greater than 40 .mu.m in diameter, using an aqueous eluent with low salt concentration. This eluent causes the hemolysis of red blood cells, which can alter the equilibrium between free and cell-bound concentrations of the drug and its metabolites.
U.S. Pat. No. 4,544,485 discloses an internal surface reverse phase (ISRP) resin, which is an HPLC chromatographic packing material useful for the analysis of hydrophobic analytes in sera and plasma by direct injection, without column deterioration due to protein accumulation. Such an ISRP resin is comprised of porous particles having hydrophobic inner pore surfaces and hydrophilic outer particle surfaces. To analyze hydrophobic drugs in blood using ISRP resin, however, prior art methods require separation of large cellular components from the sera or plasma before analysis, because the cellular components would mechanically block the chromatographic system. I. H. Hagestam et al., Anal. Chem. 57, 1757-1763 (1985); S. E. Cook et al., J. Chromatography 368, 233-346 (1986); T. C. Pinkerton et al., Biochromatography 1, 98-104 (1986); T. Nakagawa et al., J. Chromatography 420, 297-311 (1987); T. C. Pinkerton et al., J. Chromatography 367, 412-418 (1986).
Another method using ISRP resin requires dilution of the blood samples with water followed by filtration prior to injection onto the HPLC column "Pinkerton".TM. Internal Surface Reversed-Phase Separation, Application Note No. 18 (Regis Chemical Co., 8210 Austin Avenue, Morton Grove, Ill. 60053). These dilution and filtration steps are time consuming. Additionally, the filtration step removes certain whole blood components, thus hindering analysis of whole blood.
A need exists, therefore, for a rapid process for chromatographic separation of hydrophobic components from whole blood that does not require pretreatment of blood samples.